The level of creatinine in samples of physiological fluids, such as whole blood, serum, plasma or urine, is an important indicator of renal function. Creatine phosphate is stored in the muscles of vertebrates and provides an energy reserve. It is irreversibly converted into creatinine (a degradation product) and the energy rich phosphate group. During normal muscle function about 1-2% per day of the total amount of creatine phosphate is converted into creatinine. Creatinine is released into the blood and removed by the kidneys. In a healthy individual the level of creatinine is thus relatively constant at about 35 to about 75 μM. If the level of creatinine in the blood increases it may be a sign of some malfunction of the kidneys. In such cases the level of creatinine may increase to levels as high as 2,000 μM.
The level of creatinine in a sample of physiological fluid derived from a subject can be measured enzymatically, for example using creatinine iminohydrolase (by detection of NH3) or creatinine amidohydrolase. Creatinine amidohydrolase is also referred to as “creatininase”. In the case of creatininase, the creatinine level in a physiological fluid can be determined by a cascade of enzymatic reactions involving the enzymes creatininase (EC 3.5.2.10), creatine amidinohydrolase (EC 3.5.3.3—“creatinase”) and sarcosine oxidase (EC 1.5.3.1) as represented by the following reactions:

This reaction cascade results in the formation of H2O2, which in turn may be detected amperometrically or photometrically. In some systems a further enzyme (e.g., peroxidase) or an indicator may be used (e.g., a luminophor).
The level of creatinine in samples of physiological fluids can be measured using a sensor adapted for measurement of creatinine, for example a sensor comprising creatininase in combination with the enzymes creatinase and sarcosine oxidase. Such sensors are often referred to as biosensors and may employ both electrochemical and/or photometric principles.
The intermediate product creatine is also present in samples of blood, serum, plasma or urine as such. Therefore, a dual sensor system is preferably employed if the creatinine is to be determined by the above cascade of enzymatic reactions. Using a dual sensor system the creatinine may be determined as the difference between the total of the two substances and the intermediate product alone. Accordingly, in a first sensor for the determination of the total concentration of creatinine and creatine in the sample, both creatininase, creatinase and sarcosine oxidase are present for converting creatinine and creatine into H2O2. In a second sensor for the determination of the concentration of creatine in the sample, creatinase and sarcosine oxidase are present for converting creatine into H2O2. The concentration of creatinine in the sample is thus determined from the difference between the total concentration of creatinine and creatine in the sample and the concentration of creatine in the sample.
Biosensors designed for analysis of consecutive samples of physiological fluids commonly employ a flow channel for delivery of samples to and from the sensor; where delivery of each sample is typically followed by a rinse step prior to analysis of a subsequent sample. The rinse step serves to prevent contamination of one sample by the next, but may additionally be used to prevent bacterial growth and bio-film formation in the sensor and sample flow channels. Rinse (or preservative) solutions comprising the active agent 2-methyl-4-isothiazolin-3-one, also known as Methylisothiazolinone or MIT, or other isothiazolinone-derived biocides may be utilized for controlling microbial growth in water-containing solutions.
Isothiazolinone or MIT, present in preservative solutions, are thiol-interactive agents, that interact with various proteins/enzymes in bacteria and fungi, leading to inhibition of cell growth; irreversible cell damage and cell death. A drawback with use of isothiazolinone or MIT agents is that by reacting and inactivating one or more thiol group-containing enzyme in a biosensor, they can limit the useful lifetime of the biosensor. Creatinase is a thiol containing hydrolase, whose activity may be inhibited by thiol-interactive agents, such as isothiazolinone-derived agents, including MIT, which limits the use of these preservative agents in sensors used for measuring creatinine in samples of physiological fluids.
Accordingly, there exists a need for a creatinase enzyme that is more resistant to thiol-interactive agents; allowing the use of such agents in the rinse solution in a sensor for measurement of creatinine. Furthermore, there is a need for a creatinase enzyme having both enhanced resistance to thiol-interactive agents, while at the same time having thermostable properties. This is because biosensors mounted in an analyzer are commonly used and stored at 37° C., so it is important that the components of the biosensor are stable over a range of ambient temperatures, for example at least up to 37° C.
According to JPH10174585 (A), bacteria belonging to the genus Alcaligenes, in general, exhibit excellent thermal stability. JPH10174585 (A), further discloses the use of protein engineering techniques to produce a mutant Alcaligenes gene encoding a mutant creatine amidinohydrolase having improved long-term stability at temperatures such as 45° C. The mutant enzyme was characterized by one, or two or more combined substituted positions out of glutamic acid at 15th position, and arginine at 104th and 135th positions in comparison with a wild type creatine amidinohydrolase; where the wild type sequence is set out in AB016788.
An Alcaligenes strain KS-85 gene is reported to encode a thermostable creatinase (EC 3.5.3.3) in GenBank: BAA88830.1.
JPH07255485 (A) describes a mutant creatinase obtained by mutation of a Flavobacterium U-188 creatinase gene mutation; whereby at least one of the 166th, 277th and 328th amino acid sequences is substituted with a hydrophobic amino acid such as isoleucine. The mutant creatinase held activity for 1 hour when stored at 55° C.